1. Technical Field
The present invention concerns assays for determining presence, and for quantitation, of species sources of DNA. More specifically, the invention concerns assay methods for detecting and quantitating ruminant-source material in animal feed and pork material in foods. In addition, the invention extends to assay methods for detecting and quantitating beef and chicken material in foods of mixed or complex sources, and to products used in performing the foregoing methods. In addition, the invention extends to detecting and quantitating ruminant-source, pork-source, beef-source, and chicken-source material in cosmetics and other substances, which may be ingested by humans.
2. Related Art
Animal Species Involved
With the exception of the chicken assay disclosed hereinafter, all of the assays described herein are directed to hoofed mammals of the order Artiodactyla. Within that order, two suborders are of interest here: Suiforms and Ruminantia. Within Suiforms the family Suidae is of interest because it includes the common pig, Sus scrofa. The Ruminantia (“cud-chewing” animals) as a whole are of interest, because of governmental disease-control guidelines and regulations discussed hereinafter. Within Ruminantia, the family of principal interest is Bovidae, which includes cattle (cows), sheep, and goats. Within Bovidae the principal species of interest is Bos taurus, whose meat (beef) is widely consumed; the species Ovis aries is also of some interest because its meat (lamb) is also widely consumed. Deer (Odocoileus virginianus, in Cervidae family) and antelope (Antilocarpa americana in Bovidae family) are also among the Ruminantia whose meat is consumed to some extent. The assays specifically described hereinafter involve animals used as food.
Mad Cow Disease
Bovine spongiform encephalopathy (BSE), commonly referred to as “mad cow disease,” has a human form termed vCJD that is a variant of Creutzfeldt-Jakob disease, a fatal neurodegenerative disease that has caused many deaths in the United Kingdom. See P. Brown, Bovine spongiform encephalophathy and variant Creutzfeldt-Jakob disease, Br. Med. J. 322 (2001) 841-844. In response to the BSE epidemic in Europe, the United States Food and Drug Administration (FDA) imposed strict guidelines in 1997, prohibiting the use of ruminant-derived protein in the manufacture of animal feed intended for cows or other ruminants. It is widely believed that the practice of utilizing ruminant carcasses in animal feed for livestock is responsible for the spread of BSE to epidemic proportions. See P. Brown (2001), supra. As a result, the need for sensitive detection of ruminant species remains in animal feed is a paramount agricultural issue.
Pork and Beef Avoidance
The risk associated with infectious transmissible spongiform encephalopathy in humans has discouraged many individuals around the globe from consuming beef. Hindu populations also choose not to eat beef, while Jewish and Muslim populations choose to avoid consumption of pork, even in minute quantities, due to their religious beliefs. Many consumers prefer to include more chicken in their diet instead of beef or pork. In addition to concerns about infectious disease and religious concerns, many individuals are altering their eating behavior to include more chicken simply to reduce dietary fat intake in accordance with health trends. Other consumers, however, may avoid chicken because of fear of Salmonella infection. Any conceivable ambiguity in the labeling practices of commercial suppliers or grocery stores is unacceptable to these consumer subsets. The need for sensitive detection and quantitation of bovine, porcine, and chicken species in food and mixed-food products is critical in response to this consumer demand.
Prior Detection Methods
The quantitative detection of meat species sources in mixed food samples has been approached using a variety of different systems. Early approaches to identify species-specific components within mixed samples involved the use of high-performance liquid chromatography. See E. O. Espinoza, M. A. Kirms, M. S. Filipek, Identification and quantitation of source from hemoglobin of blood and blood mixtures by high performance liquid chromatography, J. Forensic Sci. 41 (1996) 804-811; H. I. Inoue, H. F. Takabe, O. Takenaka, M. Iwasa, Y. Maeno, Species identification of blood and blood-stains by high-performance liquid chromatography, Int. J. Legal Med. 104 (1990) 9-12. These methods have proven useful for the identification of many different animal species, but the detection limits using these approaches are restrictive. The detection of nuclear DNA sequences has also been useful in this regard, but is limited as a result of their generally low copy number. See R. Meyer, U. Candrian, J. Luthy, Detection of pork in heated meat products by the polymerase chain reaction, J. AOAC Int. 77 (1994) 617-622. Meat species identification using enzyme-linked immunosorbent assays, see F. C. Chen, Y. H. Hsieh, Detection of pork in heat-processed meat products by monoclonal antibody-based ELISA, J. AOAC Int. 83 (2000) 79-85, and protein profiles, see H. J. Skarpeid, K. Kvaal, K. I. Hildrum, Identification of animal species in ground meat mixtures by multivariate analysis of isoelectric focusing protein profiles, Electrophoresis 19 (1998) 3103-3109, have also been used.
But assays based on the polymerase chain reaction (PCR) are currently the method of choice for species identification. See J. H. Calvo, P. Zaragoza, R. Osta, A quick and more sensitive method to identify pork in processed and unprocessed food by PCR amplification of a new specific DNA fragment, J. Anim. Sci. 79 (2001) 2108-2112. PCR analysis of species-specific mitochondrial DNA sequences is the most common method currently used for identification of meat species in food, see B. L. Herman, Determination of the animal origin of raw food by species-specific PCR, J. Dairy Res. 68 (2001) 429-436; T. Matsunaga, K. Chikuni, R. Ranabe, S. Muroya, K. Shibata, J. Yamada, Y. Shinmura, A quick and simple method for the identification of meat species and meat products by PCR assay, Meat Sci. 51 (1999) 143-148; R. Meyer, C. Hofelein, J. Luthy, U. Candrian, Polymerase chain reaction-restriction fragment length polymorphism analysis: A simple method for species identification in food, J. AOAC Int. 78 (1995) 1542-1551; S. Lahiff, M. Glennon, L. O'Brien, J. Lyng, T. Smith, M. Maher, N. Shilton, Species-specific PCR for the identification of bovine, porcine and chicken species in meat and bone meal (IBM), Mol. Cell. Probes 15 (2001) 27-35; L. Partis, D. Croan, Z. Guo, R. Clark, T. Coldham, J. Murby, Evaluation of a DNA fingerprinting method for determining the species origin of meats, Meat Sci. 54 (2000) 11 369-376; J. F. Montiel-Sosa, E. Ruiz-Pesini, J. Montoya, P. Roncales, M. J. Lopez-Perez, A. Perez-Martos, Direct and highly species-specific detection of pork meat and fat in meat products by PCR amplification and mitochondrial DNA, J. Agric. Food Chem. 48 (2000) 2829-2832, and animal feedstuffs, see F. Bellagamba, V. M. Moretti, S. Comincini, F. Valfre, Identification of species in animal feedstuffs by polymerase chain reaction-restriction fragment length polymorphism analysis of mitochondrial DNA, J. Agric. Food Chem. 49 (2001); 3775-3781; M. Tartaglia, E. Saulle, S. Pestalozza, L. Morelli, G. Antonucci, P. A. Battaglia, Detection of bovine mitochondrial DNA in ruminant feeds: a molecular approach to test for the presence of bovine-derived materials, J. Food Prot. 61 (1998) 513-518; P. Krcmar, E. Rencova, Identification of bovine-specific DNA in feedstuffs, J. Food Prot. 64 (2001) 117-119.
The advantage of mitochondrial-based DNA analyses derives from the fact that there are many mitochondria per cell and many mitochondrial DNA molecules within each mitochondrion, making mitochondrial DNA a naturally amplified source of genetic variation. Recently, PCR-based methods have been reported that use multi-copy nuclear DNA sequences such as satellite DNA, see Z. Guoli, Z. Mingguang, Z. Zhijiang, O. Hongsheng, L. Qiang, Establishment and application of a polymerase chain reaction for the identification of beef, Meat Sci. 51 (1999) 233-236; J. H. Calvo, C. Rodellar, P. Zaragoza, R. Osta, Beef- and bovine-derived material identification in processed and unprocessed food and feed by PCR amplification, J. Agric. Food Chem. 50 (2002) 5262-5264, and repetitive elements, see J. H. Calvo, P. Zaragoza, R. Osta, A quick and more sensitive method to identify pork in processed and unprocessed food by PCR amplification of a new specific DNA fragment, J. Anim. Sci. 79 (2001) 2108-2112; K. Tajima, O. Enishi, M. Amari, M. Mitsumori, H. Kajikawa, M. Kurihara, S. Yanai, H. Matsui, H. Yasue, T. Mitsuhashi, T. Kawashima, M. Matsumoto, PCR detection of DNAs of animal origin in feed by primers based on sequences of short and long interspersed repetitive elements, Biosci. Biotechnol. Biochem. 66 (2002) 2247-2250. Like mitochondrial-based systems, these nuclear PCR-based assays take advantage of multiple target amplification sites in the genome of interest. However, many of these systems require additional procedural steps (such as endonuclease digestion) and at least 1-250 pg of starting DNA template for species detection. See J. H. Calvo, P. Zaragoza, R. Osta, A quick and more sensitive method to identify pork in processed and unprocessed food by PCR amplification of a new specific DNA fragment, J. Anim. Sci. 79 (2001) 2108-2112; J. H. Calvo, C. Rodellar, P. Zaragoza, R. Osta, Beef- and bovine-derived material identification in processed and unprocessed food and feed by PCR amplification, J. Agric. Food Chem. 50 (2002) 5262-5264. Also, Tajima and co-workers, see K. Tajima, O. Enishi, M. Amari, M. Mitsumori, H. Kajikawa, M. Kurihara, S. Yanai, H. Matsui, H. Yasue, T. Mitsuhashi, T. Kawashima, M. Matsumoto, PCR detection of DNAs of animal origin in feed by primers based on sequences of short and long interspersed repetitive elements, Biosci. Biotechnol. Biochem. 66 (2002) 2247-2250, recently reported the development of PCR assays for the detection of ruminant-, pig-, and chicken-derived materials based on sequences of short and long interspersed repetitive elements.
These assays exceed the detection limits of previously reported assays. See T. Matsunaga, K. Chikuni, R. Ranabe, S. Muroya, K. Shibata, J. Yamada, Y. Shinmura, A quick and simple method for the identification of meat species and meat products by PCR assay, Meat Sci. 51 (1999) 143-148; S. Lahiff, M. Glennon, L. O'Brien, J. Lyng, T. Smith, M. Maher, N. Shilton, Species-specific PCR for the identification of bovine, porcine and chicken species in meat and bone meal (MBM), Mol. Cell. Probes. 15 (2001) 27-35; M. Tartaglia, E. Saulle, S. Pestalozza, L. Morelli, G. Antonucci, P. A. Battaglia, Detection of bovine mitochondrial DNA in ruminant feeds: a molecular approach to test for the presence of bovine-derived materials, J. Food Prot. 61 (1998) 513-518. However, there are several limitations to their methods. Primarily, the detection of PCR products is exclusively gel based and thus non-quantitative. In addition, the relatively large size of the PCR amplicons for the assays (179-201 bp) reported by Tajima and co-workers, see K. Tajima, O. Enishi, M. Amari, M. Mitsumori, H. Kajikawa, M. Kurihara, S. Yanai, H. Matsui, H. Yasue, T. Mitsuhashi, T. Kawashima, M. Matsumoto, PCR detection of DNAs of animal origin in feed by primers based on sequences of short and long interspersed repetitive elements, Biosci. Biotechnol. Biochem. 66 (2002) 2247-2250, may limit their utility for testing trace materials that contain degraded DNA (or truncated sequences).
Cosmetics and Other Materials
Inedible remnants of cows, sheep, and other animals are rendered into fat (or “tallow”) as well as meat-and-bone meal. The fat “is used in an amazing array of products (such as soap, lipstick, linoleum, and glue).” Some of these products, such as lipstick and glue, may be ingested by human users. Considerations similar to those applying to meat may therefore apply to potentially ingested products such as lipstick that may contain ruminant-source, pork-source, or beef-source fat. Such products are thus a potential vector for BSE or religious issues. In addition, it is well known that the Sepoy Mutiny of 1857 was at least in part triggered by rumors that new British Enfield rifle cartridges were greased with animal fat from cows and pigs, thus offending both Hindus and Moslems. Hence, some consumers may wish to avoid such animal-fat products.
Short interspersed elements (SINEs) reside within almost every genome that has been studied to date. Most SINEs have amplified in the past 65 million years and are thought to have been spread throughout each genome via an RNA-mediated duplication process termed retroposition. P. L. Deininger, M. A. Batzer, Evolution of retroposons, Evol. Biol. 27 (1993) 157-196. Because each of the SINE families within the different genomes was derived independently, every mammalian order has a significant number (in excess of 100,000) of characteristic mobile elements.
PCR Terminology
In a polymerase chain reaction (PCR), a predetermined DNA sequence (of a genome) from a DNA sample is multiplied (“amplified”) many times to produce a resultant product in which the concentration of the predetermined sequence is greatly increased relative to the concentration in the original DNA sample. This process facilitates detection of the presence of the predetermined DNA sequence (also referred to hereinafter at times as the “sequence of interest”). The amplified sequence in PCR typically contains the DNA sequence of interest flanked at each end by another short sequence. The total amplified sequence is termed an amplicon. The amplicon may be represented as A-X-B-Y-C, where B=the sequence of interest, A=a flanking sequence, C=another flanking sequence, and X and Y are nucleotide sequences. Sequences complementary to A and C, i.e., A′ and C′, are known as primers; A′ is the “forward” primer and C′ is the “reverse” primer. The sequence of regions A through C is chosen so that the region B can be amplified using the primers A′ and C′ having sequences complementary to regions A and C. At times hereinafter, when the sequence of interest B is a SINE, such an amplicon A-B-C is said to be representative of SINE B.
A primer is a reagent that facilitates PCR. Primers are short segments of DNA which are complementary to the two segments of DNA within a strand of DNA that flank the DNA sequence which is to be copied from the strand of DNA (i.e., amplified); in PCR they bind (“anneal”) to the DNA sequence which is to be copied. Primers can be specific for a known DNA sequence or can be nonspecific in which case they bind to many genetic sequences. The present invention is concerned primarily with specific primers. More than one primer set can usually be designed for a given sequence of interest. Various constraints and trade-offs govern design and selection of primers.
The copy number is a measure of the number of copies of a given DNA sequence found in a given kind of DNA sample. When the copy number is high, a PCR will produce a higher concentration of the given DNA sequence than when the copy number is low. Detection of a sequence with a high copy number is therefore easier.
The size (length) of an amplicon is measured in terms of base pairs (bp). When a DNA sequence is degraded or truncated, PCR may be unsuccessful. It has been shown that selection of a smaller amplicon, if possible, helps to address this problem, since PCR product yield is inversely correlated to amplicon length. See A. K. Lindqvist, P. K. Magnusson, J. Balciuniene, C. Wadelius, E. Lindholm, M. E. Alarcon-Riquelme, U. B. Gyllensten, Chromosome-specific panels of tri- and tetranucleotide microsatellite markers for multiplex fluorescent detection and automated genotyping: evaluation of their utility in pathology. Genome Res. (1996) 6:1170-1176; A. Beckmann, U. Vogt, N. Huda, K. S. Zänker, B. H. Brandt, Direct-Double-Differential PCR for Gene Dosage Quantification of c-myc, Clin. Chem. (1999) 45:141-143.